Method of ameliorating uremia

ABSTRACT

A method of ameliorating symptoms of uremia includes administering a therapeutic agent including beraprost or each isomer constituting beraprost, or a salt thereof as an effective ingredient to an animal belonging to the Family Canidae, wherein an effective dosage is a dosage that does not significantly lower blood pressure.

TECHNICAL FIELD

This disclosure relates to a therapeutic agent and a treatment methodfor ameliorating symptoms of uremia in a patient suffering from chronicrenal failure.

BACKGROUND

Uremia is a syndrome showing various clinical symptoms stemmed fromdysfunctions of various organs and tissues, caused by the toxiccomponents in the urine accumulated in the blood. Uremia occurs whenurine cannot be excreted from the body due to failure of renal functionirrespective of the primary disease. Uremia has a wide variety ofclinical symptoms including gastrointestinal abnormalities such asanorexia, nausea, vomiting, stomatitis and enteritis; nervous systemabnormalities such as apathy, unconcern, deterioration of memory,depressed state and coma; circulatory abnormalities such as anemia,erythropoietic disorders, hypertension, ischemic heart diseases,pericarditis and myocarditis; pigmentation; itching; skin abnormalitiessuch as subcutaneous hemorrhage and so on. Uremia is caused by uremicsubstances accumulated in the blood. It is thought that there areseveral tens or more uremic substances at present, including, forexample, methylguanidine, indole compounds, malondialdehyde, creatinine,β-aminoisobutyric acid, transketolase inhibitors, polyamines, lipolyticinhibitors, urea, and phenols (see, for example, Vanholder, R. et al.,Kidney International, 63, 1934-1943, 2003).

However, it has not yet been clarified which substance, among theseuremic substances, essentially participates in the onset of uremicsymptoms. Further, although it has not been clarified whether uremia iscaused by a single substance or by compound effect of a plurality ofthese substances, it is thought that the probability of the latter ishigher. Those facts clearly indicate the difficulty in therapy of uremiaby drugs. Anyway, unless the uremic substances are excreted from thebody or unless the blood levels of the uremic substances are lowered byan appropriate means, the patient with uremia will die. The therapeuticmethods said to be effective for the treatment of uremia are kidneytransplantation, dialysis and active carbon preparation treatment. Theadvantage of the therapeutic methods common to the latter two methods isthat a plurality of uremic substances including unknown substances canbe removed or diluted nonspecifically by a physical means. However, themethods have drawbacks described below.

Active carbon preparation is orally administered. The mechanism ofaction of the active carbon preparation is based on the adsorption ofvarious uremic substances generated in digestive organs and excretionthereof from the body. Thus, although the active carbon preparation iscalled an oral drug, the therapy may be considered as a physicaltherapeutic method similar to the dialysis treatment described below.Since the active carbon preparation has drawbacks in that taking is noteasy, it has a strong tendency to induce constipation, and so on, inaddition to the limited therapeutic effect, the therapeutic method isnot satisfactory at all.

Dialysis is said to be the most effective therapeutic method for uremia.In fact, by excreting uremic substances by dialysis treatment, the deathof the patient due to the uremic substances can be avoided even if thekidney function has been abolished. However, complications by dialysistreatment have been recognized as new problems. For example,complications of long-term dialysis include anemia, renalosteodystrophy, stomach cancer, pericarditis, aluminum osteodystrophy,amyloidosis, crystal arthritis, multiple polycystic kidney and so on.There are also problems of poor QOL of the patients in social life dueto the necessity to frequently go to dialysis facilities, and high costwhich is a problem in medical economics.

A method called “internal dialysis” has been reported, in whichnonprotein nitrogen is medicinally transferred to intestinal tractwithout a physical means (JP 3-163023 A). This is a phenomenon that uponadministration of a 15-keto-16-halogen-prostaglandin E₂, serumcreatinine (Cre) and blood urea nitrogen (BUN) in the blood aretransferred to the intestinal tract together with water by enteropoolingaction (an action to accumulate water in the intestinal tract). However,since enteropooling is an action to accumulate water in the intestinaltract, it necessarily accompanies diarrhea, and the reporter also refersto this point. No matter how much the serum Cre value and BUN value inthe blood are decreased, to perform this therapy which accompanies thephysical exhaustion and the risk of dehydration due to diarrhea imposesa very heavy burden on the patients with chronic renal failure, so thatit is not acceptable at all.

Accordingly, creation of a therapeutic method and therapeutic agent foruremia could be helpful.

Although uremia occurs with the progress of renal failure, both of theseare syndromes which are separately defined as described below. Grauer,G. F., PART 5, Chapter 44, Urologic Diseases, p622, In: Small AnimalInternal Medicine (Japanese version), R. W. Nelson, C. G. Couto eds.,translation supervised by Atsuhiko HASEGAWA and Hajimu TSUJIMOTO, InterZoo Tokyo, 2001 states “a wide variety of, and sometimes confused, termsare used for expressing the renal function and its deterioration”, anddefines related terms to clearly distinguish the both. Moreparticularly, it calls attention on the difference between the bothstating “Renal failure is the state in which the renal function isdecreased and the abnormalities (azotemia and decrease in the ability toconcentrate urine) are maintained, and means the function level of theorgan rather than a specific disease”, and “Uremia indicates the factthat urine exists in the blood” (see Grauer, G. F., PART 5, Chapter 44,Urologic Diseases, p622, In: Small Animal Internal Medicine (Japaneseversion), R. W. Nelson, C. G. Couto eds., translation supervised byAtsuhiko HASEGAWA and Hajimu TSUJIMOTO, Inter Zoo Tokyo, 2001). Othertechnical books of nephrology also define and describe uremia and renalfailure as different “syndromes” and not “names of diseases” (YasushiASANO, VII Chronic Renal Failure, Nephrology (Kiyoshi KUROKAWA eds.),p345, Nankodo, Tokyo, 2001).

Renal failure is classified into two different syndromes, acute renalfailure and chronic renal failure. Acute renal failure suddenly occurs,but in most cases, its renal function impairment is reversible. In fact,effective drugs and therapeutic methods have already existed, and, inmany cases, the renal function of the patients may be restored to thenormal state by merely removing the cause. On the other hand, as forchronic renal failure, it is difficult to clearly determine the time ofonset thereof, and it slowly occurs over several months to severalyears. Further, the renal damage occurred is irreversible (see, forexample, Keizo KOIDE, 1: What is Chronic Renal Failure, Chemotherapiesof Chronic Renal Failure (Keizo KOIDE and Susumu TAKAHASHI eds.), pl,TOKYOIGAKUSHA, Tokyo, 2000), and refractory to various drugs andtherapeutic methods. Thus, therapy of uremia which occurs with theprogress of chronic renal failure is especially important.

As the results of the studies so far using nephritis model animals orrenal failure model animals, various prophylactic and therapeutic drugsfor chronic renal failure or its primary disease have been found.Various drugs are now clinically used, for example, antiplatelet drugssuch as dipyridamole, dilazep hydrochloride, trapidil and aspirin; andanticoagulants such as heparin and warfarin are used forglomerulonephritis; and thiazide diuretics, loop diuretics, angiotensinconverting enzyme (ACE) inhibitors, and calcium antagonists such asdiltiazem and verapamil are used for cases where the primary disease isessential hypertension. However, any of these is nothing more than acountermeasure which delays the progress of the state of renal failuresuch as deterioration of renal function and the like, and ameliorationor disappearance of uremia by these drugs has not been reported at all.

Among the compounds of General Formula (I), beraprost sodium disclosedin JP 1-053672 B has been reported to be effective for primary diseasesof chronic renal failure. For example, in a glomerulonephritis modelsystem, Utsunomiya et al. showed that onset of glomerulonephritis can beinhibited by administering beraprost sodium, before onset, to immunecomplex-induced glomerulonephritis mouse NZB/WF1 which spontaneouslydevelops immune complex-induced glomerulonephritis, using as an indexthe effect to decrease the urinary excretion of albumin (Utsunomiya, Y.et al., Clin. Exp. Immunol., 99, 454-460, 1995). Similarly, Kushiro etal. showed, using rat glomerulonephritis models, that onset of nephritiswas prevented by prophylactically administering beraprost sodium to therats before administering the nephritis-inducing substance, in terms ofthe effect to decrease the urinary excretion of albumin (Kushiro, M. etal., Kidney International, 53, 1314-1320, 1998). Stier et al. showedthat by administering beraprost sodium together with the supply ofsaline used as the stimulation inducing hypertension and with feeding ofa special diet to the rats which spontaneously develops hypertension,the onset of glomerulonephritis was prevented, based on the urinaryprotein excretion and an image of the tissue of glomerulus (Stier, C. T.et al., J. Cardiovascular Pharmacology, 30, 285-293, 1997). Further, ithas been reported that urinary microalbumin in patients with diabeticnephropathy was decreased by the administration of beraprost sodium(Owada, A. et al., Nephron, 92(4):788-796, 2002).

Further, there are reports about prostaglandin I₂ derivatives(hereinafter also referred to as “PGI₂ derivatives” for short) otherthan the compounds represented by General Formula (I). For example, itis known that cicaprost has an activity to reduce the renal functionimpairment in streptozotocin-induced diabetic nephropathy rats (Robles,R. G. et al., J. Hypertens. Suppl., 11, 5:S208-S209, 1993) or the renalfunction impairment induced by uninephrectomy, high sodium loading andprotein loading (Villa, E. et al., Am. J. Hypertens., 6, 253-257, 1993).Similarly, in Thy-1-induced nephritis models, urinary protein isdecreased by prophylactically administering iloprost which is a PGI₂derivative before the induction of nephritis (Poelstra, K. et al., Am.J. Pathol., 142, 441-450, 1993). Proteinuria is caused by thedeterioration of barrier function to macromolecules in the kidney.Therefore, although proteinuria is a good index for examining theglomerulus function which is an aspect of the renal function,proteinuria does not directly determine the severity of uremia. Further,although proteinuria is a good index in early stage ofglomerulonephritis, diabetic nephropathy and the like, it is no longer agood index in chronic renal failure because the filtering functions tolow molecular substances are decreased in chronic renal failure.Further, all of the above-described reports show the prophylacticeffects of PGI₂ derivatives because the PGI₂ derivatives wereadministered before the onset of nephritis, and do not show thetherapeutic effect. Still further, although the reports refer to renalfunction, they do not refer to or suggest amelioration of uremia at all.

As for the effects of the compounds represented by General Formula (I),there is a report in which rat renal failure models whose primarydisease was the nephritis, prepared by administering an antibody toglomerular basement membrane were used (WO 00/067748). In the report, itwas shown that by administering a compound of General Formula (I)recited herein after observing renal failure defined by highercreatinine and BUN, increase in the renal failure markers such as amountof urinary protein excretion, serum Cre value and BUN value was reducedwhen compared with a control group. Further, it has been reported thatclinical administration of beraprost sodium reduced the decrease inrenal function in the renal failure in conservative stage, which isindicated by decrease in the creatinine clearance or in the reciprocalof serum creatinine in patients with renal failure (Fujita, T. et al.,Prostaglandins Leukot. Essent. Fatty Acids, October; 65(4): 223-227.2001). However, these reports do not contain any reference or suggestionabout uremia or uremic symptoms.

Renal diseases include hemolytic uremic syndrome (HUS) caused bythrombotic microangiopathy. HUS literally shows uremia as its clinicalsymptom. Series et al. reported that, in one case of HUS, administrationof iloprost which is one of the PGI₂ derivatives was effective for theimprovement of the serum Cre value which is a marker of renal failure(Series, C. et al., Rev. Med. Interne., 17, 76-78, 1996). However, HUSis a syndrome stemmed from the above-described easily recoverable acuterenal failure which is a pathological condition utterly different fromuremia in the patients with chronic renal failure whose body homeostasisis drastically impaired, in the resistance to drugs and therapeuticmethods. Thus, this report is also totally silent about theapplicability to the therapy of uremia in patients with chronic renalfailure, which is a severer syndrome.

On the other hand, Siegler et al. reported that PGI₂ does not have anactivity to inhibit the progress of HUS (Siegler, R. L. et al., Nephron,92, 363-368, 2002). Even about in vitro studies, there is a report whichreports that the ability of vascular endothelial cells to produce PGI₂said to be related to onset of HUS is decreased (Mitra, D. et al.,Blood, 89, 1224-1234, 1997), and a report which reports that no changewas observed on the ability to produce PGI₂ (Adler, S. and Bollu, R.Kidney & Blood Pressure Research, 21, 13-21, 1998). That is, thepharmacological effect of PGI₂ derivatives to HUS is still very unclear,and the above-mentioned Series et al. described that the above-mentionedone case was a very rare case, the relationship between HUS and PGI₂ wasstill contradicting, and frankly concluded that further study wasnecessary. Anyway, even in HUS which has a better ability to recover thebody homeostasis, the involvement of PGI₂ is still unclear, and theeffectiveness of PGI₂ to the uremia of patients with chronic renalfailure, whose ability to recover the body homeostasis is much poorer orwho have no such an ability, is not expected at all.

As described above, deterioration or amelioration of uremic symptoms inthe patients with chronic renal failure by the compounds represented byGeneral Formula (I), or even by PGI₂ derivatives including those otherthan the compounds represented by General Formula (I), has not beendescribed at all.

On the other hand, in recent years, increase in the number of patientswith chronic renal failure has become a big problem not only in humanmedicine but also in veterinary medicine. Pet animals such as dogs andcats can now receive highly nutrious diets and high veterinary services.As a result, pet animals came to have a long life similar to humans, andthe number of patients suffering from an aging-associated disease orchronic disease which is difficult to cure are now being drasticallyincreased. Chronic renal failure is observed in many kinds of petanimals. Taking cat as an example, comparisons between the chronic renalfailure of cats and humans are as follows:

In cats, chronic renal failure occurs at an especially high rate (see,for example, Dibartola, S. P. et al., J. Am. Vet. Med. Assoc., 190,1196-1202, 1987). In cats of not younger than 15 years old, chronicrenal failure amounts to as much as 30% of total diseases, and is amajor cause of death of cats (see, for example, Lulich, J. et al., TheCompendium Continuing Education, 14, 127-152, 1992). Further, in verymany cases, when a patient cat is carried in an animal hospital, the catis in considerably progressed state of chronic renal failure, andaccompanies uremia.

In general, chronic renal failure of cats is diagnosed by detecting highvalues of serum creatinine (Cre) and blood urea nitrogen (BUN) valueswhich are clinical markers of renal function, in addition to the pasthistory obtained by hearing and the clinical observations. Abnormal Creand BUN values are not detected unless 75% of nephron in both kidneyslost its function, and there is a correlation between the magnitude ofthe abnormal values of the markers of renal function and the ratio ofremaining renal function, as in the case of humans.

Uremic symptoms of cats are expressed by nausea, vomiting, diarrhea,anorexia, weight loss, decreased activity, polydipsia and diuresis,intraoral ulcer and so on. Anorexia and shortage of water intake due tothe intraoral ulcer also cause dehydration and constipation. Afterfurther progress of the diseased state, in addition to anemia,erythropoietic disorders, loss of appetite and depressed state,encephalopathy and neuropathy due to the urine toxin are observed. Asdescribed above, the progress of the diseased state of the chronic renalfailure and the diversity of the accompanied uremic symptoms are thesame as in humans.

Since the cats having progressed uremia tend to develop dehydration, assymptomatic treatments, hydration, and fluid replacement by intravenousor subcutaneous drip infusion for the purpose of dilution of the uremicsubstances are also performed. For anemia in cats with uremia,erythropoietin having hematopoietic activity is also administered. Sinceappetite is often reduced also by anemia, an appetite-stimulatingtreatment is also performed in parallel. For the amelioration ofsymptoms such as vomiting, diarrhea and stomatitis, symptomatictreatment drugs corresponding to the symptoms are also often used. Theindividual symptomatic treatments selected for these uremic symptoms arealso the same as in the case of human patients with uremia. There is adifference in drug metabolism between cats and humans. For example,acetoaminophene which can be safely used in humans induces a seriousside effect (acetoaminophene poisoning) in cats. In addition to this, anumber of antibiotics and anti-inflammatory agents are listed as drugsrequiring caution. Further, since the homeostatic functions in the catsshowing uremic symptoms are deteriorated, much caution and care areneeded for the usage and dosage of the drugs.

Similar to humans, active carbon preparation is used for the therapy ofuremia in cats and dogs. The name of the drug for humans is Kremezin andthe name of the animal drug is Covalzin. In this therapy, thepreparation is orally administered daily, and the substances causinguremia are made to be adsorbed thereto in the digestive tract, and areexcreted together with feces. However, similar to humans, taking is noteasy and reduction of appetite and constipation are also induced.Further, there is a concern that useful substances may be removed by thenonspecific adsorption of substances.

Similar to humans, dialysis is a very effective therapy of uremia incats. However, since it is expensive, the therapy has not prevailed to ageneral therapy. Although kidney transplantation may also be performedin cats as a curative treatment for chronic renal failure and uremia,this has also scarcely spread because it is expensive.

As described above, uremia of animals and humans are similar in variousaspects including the state of disease of chronic renal failure,clinical symptoms of accompanying uremia, therapeutic methods,symptomatic treatments and so on. Further, since there are problems intherapeutic methods as in humans, creation of an excellent therapeuticmethod and therapeutic drug for uremia are demanded by the clients(owner) of the patient animals and veterinarians.

It could therefore be helpful to provide a therapeutic agent and atreatment method for uremia of humans and animals, which therapeuticagent can be easily taken, has lower side effects, and which is notexpensive in view of medical economics.

SUMMARY

One of the reasons why the creation of therapeutic agent and therapeuticmethod of ameliorating uremia has not been achieved in spite of thestrong demand is that an appropriate experimental model of uremia hasnot been established. After a series of investigations and studies, wefocused our attention on the fact that patient cats suffering fromchronic renal failure accompanying uremia at a high rate come to animalhospitals, and a number of patient cats die because they cannot receivedialysis treatment because of economical or technical reason. In view ofthis, after intensive studies on patient cats, we discovered thatimprovement in renal function is not directly linked to the ameliorationof uremia, and the drugs comprising as an effective ingredient acompound represented by General Formula (I) described below areeffective for the therapy of uremia, based on the discovery that uremiamay be ameliorated or disappear even under the state that the renalfailure markers are continuously increased.

We thus provide:

1. A therapeutic agent for uremia in patients suffering from chronicrenal failure, the therapeutic agent comprising as an effectiveingredient a compound of General Formula (I):

wherein R¹ is(A) COOR², wherein R² is1) hydrogen or a pharmaceutically acceptable cation,2) C₁-C₁₂ straight alkyl or C₃-C₁₄ branched alkyl,3) —Z—R³, wherein Z is covalent bond, or straight or branched alkylenerepresented by C_(t)H_(2t) wherein t is an integer of 1 to 6, R³ isC₃-C₁₂ cycloalkyl or C₃-C₁₂ cycloalkyl substituted with 1 to 3 R⁴(s)wherein R⁴ is hydrogen or C₁-C₅ alkyl,4) —(CH₂CH₂O)—CH₃, wherein n is an integer of 1 to 5,5) —Z—Ar¹, wherein Z represents the same meanings as described above,Ar¹ is phenyl, α-naphthyl, β-naphthyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,α-furyl, β-furyl, α-thienyl, β-thienyl or substituted phenyl (whereinthe substituent(s) is(are) at least one of chlorine, bromine, fluorine,iodine, trifluoromethyl, C₁-C₄ alkyl, nitro, cyano, methoxy, phenyl,phenoxy, p-acetamidebenzamide, —CH═N—NH—C(═O)—NH₂, —NH—C(═O)-Ph,—NH—C(═O)—CH₃ and —NH—C(═O)—NH₂),6) —C_(t)H_(2t)COOR⁴, wherein C_(t)H_(2t) and R⁴ represent the samemeanings as described above,7) —C_(t)H_(2t)N(R⁴)₂, wherein C_(t)H_(2t) and R⁴ represent the samemeanings as described above,8) —CH(R⁵)—C—(═O)—R⁶, wherein R⁵ is hydrogen or benzoyl, and R⁶ isphenyl, p-bromophenyl, p-chlorophenyl, p-biphenyl, p-nitrophenyl,p-benzamidephenyl or 2-naphthyl,9) —C_(p)H_(2p)—W—R⁷, wherein W is —CH═CH—, —CH═CR⁷— or —C≡C—, whereinR⁷ is hydrogen, C₁-C₃₀ straight or branched alkyl or C₇-C₃₀ aralkyl, pis an integer of 1 to 5, or10) —CH(CH₂OR⁸)₂, wherein R⁸ is C₁-C₃₀ alkyl or acyl,

(B) —CH₂OH,

(C) —C(═O)N(R⁹)₂,wherein R⁹ is hydrogen, C₁-C₁₂ straight alkyl, C₃-C₁₂ branched alkyl,C₃-C₁₂ cycloalkyl, C₄-C₁₃ cycloalkylalkylene, phenyl, substituted phenyl(wherein the definition(s) of the substituent(s) is(are) the same asthose described in (A) 5) mentioned above), C₇-C₁₂ aralkyl or —SO₂R¹⁰wherein R¹⁰ is C₁-C₁₀ alkyl, C₃-C₁₂ cycloalkyl, phenyl, substitutedphenyl (wherein the definition(s) of the substituent(s) is(are) the sameas those described in (A) 5) mentioned above), or C₇-C₁₂ aralkyl,wherein the two R⁹s may be the same or different, with the proviso thatwhen one of them is —SO₂R¹⁰, the other R⁹ is not —SO₂R¹⁰, or(D) —CH₂OTHP (wherein THP is tetrahydropyranyl),

A is

1) —(CH₂)_(m)—,

2) —CH═CH—CH₂—, 3) —CH₂—CH═CH—,

4) —CH₂—O—CH₂—,

5) —CH═CH—, 6) —O—CH₂— or

7) —C≡C—, wherein m is an integer of 1 to 3,Y is hydrogen, C₁-C₄ alkyl, chlorine, bromine, fluorine, formyl, methoxyor nitro,B is —X—C(R¹¹)(R¹²)OR¹³, wherein R^(H) is hydrogen or C₁-C₄ alkyl, R¹³is hydrogen, C₁-C₁₄ acyl, C₆-C₁₅ aroyl, tetrahydropyranyl,tetrahydrofuranyl, 1-ethoxyethyl or t-butyl,

X is

1) —CH₂—CH₂—

2) —CH═CH— or 3) —C≡C—, R¹² is

1) C₁-C₁₂ straight alkyl, C₃-C₁₄ branched alkyl,2) —Z—Ar², wherein Z represents the same meanings as described above,Ar^(e) is phenyl, a-naphthyl, β-naphthyl, or phenyl substituted with atleast one substituent selected from the group consisting of chlorine,bromine, fluorine, iodine, trifluoromethyl, C₁-C₄ alkyl, nitro, cyano,methoxy, phenyl and phenoxy,3) —C_(t)H_(2t)OR¹⁴, wherein C_(t)H_(2t) represents the same meanings asdescribed above, R¹⁴ is C₁-C₆ straight alkyl, C₃-C₆ branched alkyl,phenyl, phenyl substituted with at least one substituent selected fromthe group consisting of chlorine, bromine, fluorine, iodine,trifluoromethyl, alkyl, nitro, cyano, methoxy, phenyl and phenoxy,cyclopentyl, cyclohexyl, cyclopentyl substituted with 1 to 4 C₁-C₄straight alkyl or cyclohexyl substituted with 1 to 4 C₁-C₄ straightalkyl,4) —Z—R³, wherein Z and R³ represent the same meanings as mentionedabove,5) —C_(t)H_(2t)—CH═C(R¹⁵)R¹⁶, wherein C_(t)H_(2t) represents the samemeanings as mentioned above, R¹⁵ and R¹⁶ represent hydrogen, methyl,ethyl, propyl or butyl, or6) —C_(u)H_(2u)—C≡C—R¹⁷, wherein u is an integer of 1 to 7, C_(u)H_(2u)is straight or branched alkylene, and R¹⁷ is C₁-C₆ straight alkyl,E is hydrogen or —OR¹⁸, wherein R¹⁵ is C₁-C₁₂ acyl, C₇-C₁₅ aroyl or R²(wherein R² represents the same meanings as described above),the formula includes d-isomers, 1-isomers and racemic compounds.

2. The therapeutic agent for uremia in patients suffering from chronicrenal failure, according to item 1, wherein in General Formula (I),

R¹ is COOR², wherein R² is hydrogen or a pharmaceutically acceptablecation,A is —(CH₂)_(m)—, wherein m is an integer of 1 to 3,Y is hydrogen,B is —X—C(R¹¹)(R¹²)OR¹³, wherein R¹¹ and R¹³ are hydrogen,

X is —CH═CH—,

R¹² is —C_(u)H_(2u)—C≡C—R¹⁷ wherein u is an integer of 1 to 7,C_(u)H_(2u) is straight or branched alkylene, and R¹⁷ is C₁-C₆ straightalkyl, andE is hydrogen or —OR² (wherein R² represents the same meanings asdescribed above).

3. The therapeutic agent for uremia in patients suffering from chronicrenal failure, according to item 1, wherein in General Formula (I),

R¹ is COOR², wherein R² is hydrogen or sodium ion,A is —(CH₂)_(m)—, wherein m is 3,Y is hydrogen,B is —X—C(R¹¹)(R¹²)OR¹³, wherein R¹¹ and R¹³ are hydrogen,

X is —CH═CH—,

R¹² is —C_(u)H_(2u)—C≡C—R¹⁷ wherein u is 3, C_(u)H_(2u) is branchedalkylene, and R¹⁷ is methyl, and

E is —OH.

4. A treatment method of ameliorating symptoms of uremia in a patientsuffering from chronic renal failure, the method comprisingadministering the therapeutic agent for uremia according to any one ofitems 1 to 3 to the patient.

5. Use of the compound represented by General Formula (I) recited in anyone of items 1 to 3, for the production of a therapeutic agent foruremia in patients suffering from chronic renal failure.

We provide a method of ameliorating symptoms of uremia includingadministering a therapeutic agent including beraprost or each isomerconstituting beraprost, or a salt thereof as an effective ingredient toan animal belonging to the Family Canidae, wherein an effective dosageis a dosage that does not significantly lower blood pressure.

The therapeutic agent for uremia in patients with chronic renal failure,comprising a compound represented by General Formula (I) ameliorates theuremia concurred in patients with chronic renal failure without a sideeffect, and recovery of reduced appetite, amelioration of activity,increase in weight, and so on are achieved. Further, since the therapyis not by means of physical adsorption of uremic substances andexcretion thereof, we provide a therapeutic agent and a treatmentmethod, which agent can be easily taken, and that are not expensive inview of medical economics. The desired effect is clear alleviation ordisappearance of symptoms of uremia grasped as clinical symptoms,observed in the state of renal failure, especially even in spite of thestate wherein the decrease in renal function is progressed. Theprominent effects observed in cats which are a suitable model forevaluation of uremia are comparable to the therapeutic effects obtainedby dialysis treatment in human patients with uremia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a and 1b show the action of beraprost sodium on the heart rate ofhealthy cats. Beraprost sodium was orally administered at the doseindicated in the figures. Significant test was carried out by the t-test(paired). * p<0.05, ** p<0.01. In FIG. 1a , the ordinate in the figureindicates the measured heart rate. The abscissa in the figure indicatesthe time after administration of beraprost sodium. In FIG. 1b , theordinate in the figure indicates the relative value taking the heartrate before the administration of beraprost sodium as 100% after addingthereto the % change after administration of beraprost sodium. Theabscissa in the figure indicates the time after administration ofberaprost sodium.

FIGS. 2a-2c show the action of beraprost sodium on the blood pressure(measured value) of healthy cats. Beraprost sodium was orallyadministered at the dose indicated in the figures. In FIG. 2a , theaction of beraprost sodium on systolic pressure is shown. In FIG. 2b ,the action of beraprost sodium on diastolic pressure is shown. In FIG.2c , the action of beraprost sodium on pulse pressure is shown.

FIGS. 3a-3c show the results of administration of beraprost sodium to acat with chronic renal failure (Case-A, administration of beraprostsodium alone). In FIG. 3a , the open bar indicates the dosing period inwhich beraprost sodium alone was administered. After termination of theadministration, no treatment was performed. In the 6th month aftertermination of the administration of beraprost sodium too, tests werecarried out. As a result, uremic symptoms were not observed at all, andthe markers of renal function remained within the normal ranges, so thatchronic renal failure was cured by the administration of beraprostsodium alone. In FIG. 3a , effects of amelioration of uremic symptoms(clinical symptoms) by the administration of beraprost sodium alone tothe cat (Case-A) with chronic renal failure are shown. The abscissaindicates the number of months after administration of beraprost sodium.In FIG. 3b , the change in BUN value which is a marker of renalfunction, by the administration of beraprost sodium is shown. In FIG. 3c, the change in serum Cre value which is a marker of renal function, bythe administration of beraprost sodium is shown.

FIGS. 4a-4c show the results of administration of beraprost sodium to acat with chronic renal failure (Case-B, administration of beraprostsodium alone). In FIG. 4a , effects of amelioration of uremic symptoms(clinical symptoms) by the administration of beraprost sodium alone tothe cat (Case-B) with chronic renal failure are shown. The abscissaindicates the number of months after administration of beraprost sodium.In FIG. 4a , the open bar indicates the dosing period in which beraprostsodium alone was administered. In FIG. 4b , the change in BUN valuewhich is a marker of renal function, by the administration of beraprostsodium is shown. In FIG. 4c , the change in serum Cre value which is amarker of renal function, by the administration of beraprost sodium isshown.

FIGS. 5a-5c show the results of administration of beraprost sodium to acat with chronic renal failure (Case-C, administration of beraprostsodium alone). In FIG. 5a , effects of amelioration of uremic symptoms(clinical symptoms) by the administration of beraprost sodium alone tothe cat (Case-C) with chronic renal failure are shown. The abscissaindicates the number of months after administration of beraprost sodium.In FIG. 5b , the change in BUN value which is a marker of renalfunction, by the administration of beraprost sodium is shown. In FIG. 5c, the change in serum Cre value which is a marker of renal function, bythe administration of beraprost sodium is shown.

FIGS. 6a-6c show the results of administration of beraprost sodium to acat with chronic renal failure (Case-D, administration of beraprostsodium alone). In FIG. 6a , the open bar indicates the dosing period inwhich beraprost sodium alone was administered. In FIG. 6a , effects ofamelioration of uremic symptoms (clinical symptoms) by theadministration of beraprost sodium alone to the cat (Case-D) withchronic renal failure are shown. The abscissa indicates the number ofmonths after administration of beraprost sodium. In FIG. 6b , the changein BUN value which is a marker of renal function, by the administrationof beraprost sodium is shown. In FIG. 6c , the change in serum Cre valuewhich is a marker of renal function, by the administration of beraprostsodium is shown.

FIGS. 7a-7c show the results of administration of beraprost sodium to acat with chronic renal failure (Case-E, combination therapy). In FIG. 7a, the filled bar indicates the period in which treatments with an activecarbon preparation, calcium carbonate preparation and k/d meal werecombined with the treatment of beraprost sodium. The open bar indicatesthe period in which administration of the active carbon preparation wasnot performed because the constipation became severer. In FIG. 7a ,effects of amelioration of uremic symptoms (clinical symptoms) by theadministration of beraprost sodium alone to the cat (Case-E) withchronic renal failure are shown. The abscissa indicates the number ofmonths after administration of beraprost sodium. In this case, inaddition to the feeding of the formula meal, treatments with the activecarbon preparation and calcium carbonate preparation were combined up toan intermediate time point, and thereafter (from 4th month), only theadministration of the active carbon preparation was stopped because ofsevere constipation. In FIG. 7b , the change in BUN value which is amarker of renal function, by the administration of beraprost sodium isshown. In FIG. 7c , the change in serum Cre value which is a marker ofrenal function, by the administration of beraprost sodium is shown.

FIGS. 8a-8c show the results of administration of beraprost sodium to acat with chronic renal failure (Case-F, combination therapy). In FIG. 8a, the dotted bar indicates the period in which treatments with insulin,an active carbon preparation and a calcium carbonate preparation werecombined, and the filled bar indicates the period in which the treatmentwith beraprost sodium was additionally combined. During the two monthsbefore the start of the administration of beraprost sodium, ameliorationof uremic symptoms was not observed, but after the start of theadministration of beraprost sodium, uremia was quickly improved, and theimproved condition was continued. During this period, Cre which is amarker of renal failure continuously increased. In FIG. 8a , effects ofamelioration of uremic symptoms (clinical symptoms) by theadministration of beraprost sodium alone to the cat (Case-F) withchronic renal failure are shown. The abscissa indicates the number ofmonths after administration of beraprost sodium. In FIG. 8b , the changein BUN value which is a marker of renal function, by the administrationof beraprost sodium is shown. In FIG. 8c , the change in serum Cre valuewhich is a marker of renal function, by the administration of beraprostsodium is shown.

FIGS. 9a-9c show the results of administration of beraprost sodium to adog with chronic renal failure (Case-H, administration of beraprostsodium alone). In the figures, the dotted bar indicates the treatmentperiod in which an active carbon preparation (Covalzin) alone wasadministered, and the open bar indicates the treatment period afterswitching to the administration of beraprost sodium alone. Ameliorationof uremic symptoms during the two months in which Covalzin alone wasadministered was hardly observed, but after the start of administrationof beraprost sodium, the uremic symptoms were quickly remitted andameliorated, and the clinical score reached the full points. However,during this period, Cre which is a marker of renal failure continuouslyincreased. During period of the administration of beraprost sodium, noadverse action causing diarrhea, vomiting or the like was observed atall. In FIG. 9a , effects of amelioration of uremic symptoms (clinicalsymptoms) by the administration of beraprost sodium alone to the dog(Case-H) with chronic renal failure are shown. The abscissa indicatesthe observation period (number of months). In FIG. 9b , the change inBUN value which is a marker of renal function, by the administration ofberaprost sodium is shown. In FIG. 9c , the change in serum Cre valuewhich is a marker of renal function, by the administration of beraprostsodium is shown.

DETAILED DESCRIPTION

In General Formula (I) representing the compounds used as an effectiveingredient of the therapeutic agent,

R¹ is preferably COOH, COONa or COOMe, especially preferably COONa;A is preferably —(CH₂)₂—, —(CH₂)₃—, —CH═CH— or —O—CH₂—, especiallypreferably —(CH₂)₃—;Y is especially preferably hydrogen;B is represented by —X—C(R¹¹)(R¹²)OR¹³ wherein R¹¹ and R¹³ areespecially preferably hydrogen,X is preferably —CH═CH—, especially preferably trans —CH═CH—, R¹² ispreferably hexyl, pentyl, 1-methylpropyl, 2-chlorophenyl,propyloxymethyl, cyclohexyl, 4-hexyn-2-yl, 2-methyl-4-hexyn-2-yl, andespecially preferably 4-hexyn-2-yl; andE is especially preferably —OH.

Specific examples of the preferred compounds represented by GeneralFormula (I) include16-methyl-18,18,19,19-tetrahydro-5,6,7-trinor-4,8-inter-m-phenylene PGI₂(general name: beraprost),16-methyl-18,18,19,19-tetrahydro-5,6,7-trinor-4,8-inter-m-phenylene PGI₂sodium salt (Sodiumrac-(1R,2R,3aS,8bS)-2,3,3a,8b-tetrahydro-2-hydroxy-1-[(E)-(3S,4RS)-3-hydroxy-4-methyloct-1-en-6-ynyl]-1H-cyclopenta[b][1]benzofuran-5-butanoate:general name: beraprost sodium) and the like, and among these, beraprostsodium is preferred. However, these are nothing more than specificexamples, and the compounds are not restricted thereto.

The compounds represented by General Formula (I), especially beraprostsodium, are stable for a long time, and bioavailability thereof whenorally administered is high. Therefore, they may be especiallypreferably used because a long-term taking is required for the patientswith renal disease, especially patients with chronic renal failure.

The compounds per se represented by General Formula (I) are known, andcan be produced by known methods described in, for example, JP 1-53672B, JP 7-5582 B, JP 3-7275 A, JP 6-62599 B and so on.

The compounds represented by General Formula (I) may be usedindividually or two or more of these may be used in combination.

The term “chronic renal failure” means that the abnormal state of renalfunction wherein the number of functional nephron is decreased so thatthe excretion of nitrogen metabolites is insufficient and homeostasis ofinternal environment of the body cannot be maintained is continued for along period of time. Specifically, it may be defined as the state orsyndrome wherein blood urea nitrogen (BUN) and/or serum creatinine (Cre)value is(are) continuously elevated. This definition is essentially thesame as the definition described in the text books mentioned in thesection of Background Art. More specifically, if the serum creatininevalue measured by the enzyme method (Tomoko OHARA, Tadashi KAWAI, Kidneyand Dialysis, Vol. 39, 1995, 10) is not less than 1.4 mg/dL, the casecan be judged as chronic renal failure. However, when the once elevatedCre value is lowered by an existing therapy such as dialysis treatmentor the like, the case may be judged as chronic renal failure even if theCre value is lower than the value mentioned above.

Examples of the primary diseases of the chronic renal failure includerenal calculus, urinary obstruction, diabetic nephropathy, acuteglomerulonephritis, chronic glomerulonephritis, nephrotic syndrome,polycystic kidney, nephropathy due to infection, lupus nephritis,interstitial nephritis, acute tubulointerstitial nephritis, chronictubulointerstitial nephritis, liver cirrhosis, hepatic edema andcongestive heart failure. Our method is also effective for uremia whoseprimary disease is minor glomerular alterating nephritis, focal andsegmental glomerulonephritis, diffuse glomerulonephriti s, mesangialproliferative glomerulonephriti s, diffuse intracanalicular productivenephritis, crescentic nephritis, diffuse sclerotic glomerulonephritis orIgA nephropathy.

The term “uremia” means the state or syndrome which shows at least oneof nausea, vomiting, anorexia, loss of appetite, weight loss, decreasedactivity, diarrhea, polydipsia and diuresis, intraoral ulcer,gastrointestinal disorder, urinary retention, cranial neuropathy,circulatory abnormalities, anemia, erythropoietic disorders, skinmanifestations and ocular manifestations, caused by the fact that wastescannot be removed from the blood due to the progress of renal failure.Some text books more simply define uremia as “the condition in whichurine exists in the blood”, and a separate name “uremic syndromes” isapplied to the above-mentioned various symptoms, and the term “uremia”includes the various symptoms defined by the uremic syndromes. Examplesof the specific syndromes of uremia include encephalopathy andneuropathy due to the urine toxin, in addition to nausea, vomiting,anorexia, weight loss, decreased activity, polydipsia and diuresis,intraoral ulcer, anemia, erythropoietic disorders, loss of appetite anddepressed state, and uremia can be grasped by one or more of thesesyndromes. Thus, the therapeutic agent provides a therapeutic agent foruremia in the patients with chronic renal failure as defined above.

In the evaluation of the effect of the therapeutic agent, the point towhich the greatest attention should be paid is not to be sticked to thechange of serum Cre value and BUN value. These values are nothing morethan markers of renal failure, especially the markers of the filteringfunction of the kidney for low molecular substances, and they do notreflect the severity of uremia. In fact, by administering a compound ofGeneral Formula (I), uremic symptoms per se disappear or are amelioratedeven under the condition where the renal function is continuouslydecreased. This response is especially clearly observed in theindividuals who have a serum Cre value of not less than 2 mg/dL at thefirst visit. On the other hand, by the detoxification or the removal ofthe uremic substances by the compound of General Formula (I), althoughthe mechanism is unclear, organic regeneration of the kidney occurs andthe patient may be freed from renal failure per se. Therefore,monitoring serum Cre value and BUN value during the treatment period ismeaningful from this viewpoint.

The patient for whom the therapeutic agent is effective is notrestricted, and any subject may be treated as long as the patient isjudged to suffer from uremia due to chronic renal failure. The patientis preferably a mammal, more preferably a cat, dog or human. Amongthese, the therapeutic agent shows prominent effects in cats in view ofthe fact that when beraprost sodium included in General Formula (I) wasadministered as a therapeutic agent, uremia was ameliorated and sideeffects such as diarrhea and vomiting were not observed at all duringthe long dosing period, and that for a cat with light chronic renalfailure at a degree of uremia, needless to say amelioration of theclinical symptoms of uremia, the serum Cre value and BUN value which arerenal failure markers drastically decreased and normalized, and thenormalized state was continued for a long time so that restitutioneffect from the chronic renal failure per se was observed. Thisdiscovery is very surprising in view of the fact that chronic renalfailure is an irreversible syndrome and the effects of the existingtherapeutic drugs are nothing more than delaying the progress of chronicrenal failure. In fact, in this particular case, even after a long timefrom the end of the period of 6 months in which the test compound wasadministered, the recovered renal function continued to keep the normalvalues, and the cat was also very healthy when judged from generalconditions. The significance of the fact that these findings wereobtained not on renal failure model animals, but on actual cases ofuremia in patient cats which spontaneously developed chronic renalfailure is very great.

The dose per administration of the compound represented by GeneralFormula (I), which is an effective ingredient of the therapeutic agentis usually 0.01 μg/kg to 600 mg/kg, preferably 0.01 μg/kg to 60 mg/kg,still more preferably 0.01 μg/kg to 10 mg/kg, still more preferably 0.03μg/kg to 3 mg/kg, most preferably 0.1 μg/kg to 1 mg/kg. This dose ispreferably administered one to four times a day chronically for not lessthan 7 days, preferably not less than 30 days, but the way ofadministration is not restricted thereto.

The pharmaceutical composition can have various dosage forms. Morespecifically, in case of oral administration, the composition may be inthe form of tablets, powders, fine granules, granules, tablets, liquids,syrups, capsules, balls and sprays. Further, shaped products may becoated with a film, coated with sugar or filled in capsules. Preferredexamples include tablets, powders, fine granules, granules, tablets,liquids, syrups and capsules. The composition may be in the form ofsterilized solution or the like and may be parenterally administered.Other solute(s), such as sodium chloride and glucose in an amountsufficient to make the liquid isotonic may also be used.

In addition to the above-mentioned oral preparations, the therapeuticand prophylactic agent may be used in the form of various injectionsolutions and suppositories. Depending on the characteristics of therespective drugs, release controls such as those attaining sustainedrelease or delayed release may be applied. For example, sustainedreleasing function may be given to the drug by a known method, and apump for sustained-releasing (for example, Alzet minipump) may also beemployed, so that a wide variety administration methods may also beemployed for parenteral administration. Further, since renal diseasesare chronic diseases requiring a long-term therapy, the therapeuticagent may be preliminarily added to the formula meals for the patientswith a renal disease under the guidance of a physician or aveterinarian. The therapeutic agent may be orally administeredsubsequently to, or simultaneously with, or after an interval from theadministration of an active carbon preparation which is an existingtherapeutic agent for uremia. When the active carbon preparation isadministered subsequently to the therapeutic agent, and when thetherapeutic agent is administered for a prescribed period prior to theadministration of the active carbon preparation, the change from thetherapeutic agent to the active carbon preparation can be attainedalmost instantly. On the other hand, when the active carbon preparationis switched to the therapeutic agent, an appropriate washout period ofthe active carbon preparation is preferably given because there is apossibility that the compound represented by General Formula (I) may beadsorbed to the active carbon and its pharmacological effect may beinfluenced.

The drug having the effect to ameliorate uremia may be administeredtogether with the above-described various drugs used for amelioratingrenal failure, including angiotensin converting enzyme inhibitors andATII receptor blockers, as well as antihypertensive drugs such ascalcium blockers and β blockers, or a mixture of the therapeutic agentwith these drug(s) may be prepared and administered.

We also disclose a treatment method for uremic symptoms in the patientswith chronic renal failure. In humans, what can be treated by the methodare patients with chronic renal failure in whom uremia is complicated.The method may be applied not only to, needless to say, the patientswith uremia before receiving dialysis treatment, but also to thepatients with uremia who are receiving dialysis treatment. Further, themethod may be applied to the patients who developed a complication dueto the dialysis treatment, and to the patients who developed sideeffects by the active carbon preparation. Still further, the method maybe employed together with a drug(s) for symptomatic treatment forcomplications due to dialysis, such as erythropoietin for anemia orerythropoietic disorders. Similarly, our method may be applied topatients with uremia caused by chronic renal failure, who are receivingtherapy with active carbon preparation. It should be understood,however, that active carbon has an ability to nonspecifically adsorbsubstances, and the treatment is preferably performed taking the timingof administration of the both drugs into consideration. When the sideeffects by the active carbon preparation have been developed, thetherapy with active carbon preparation may be stopped and the therapymay be switched to our method. Uremia of pet animals other than cats anddogs may also be treated in the same way. The pet animals are notrestricted, and any of the animals which are the subjects of veterinarymedicine, for which diagnosis and therapy are demanded by the clients(owners), and which are judged as uremia caused by chronic renal failureby veterinarians may be treated, and mammals are preferably employed.Animals are classified into families, genera and species in taxonomy,and our method may be equally applied to any of them. For example, asfor cats, although domestic cats (Felis catus) are usual, other animalsbelonging to Family Falidae, genus Felis may also be preferablyemployed. In addition, animals belonging to other genera in FamilyFelidae, such as genera Panthera, Acinonyx, Neofelis and Lynx may alsobe employed. Needless to say, the method may also be preferably appliedto the animals belonging to the Family Canidae, which are pet animalsother than cats and which develop uremia caused by chronic renalfailure. Further, needless to say, uremia caused by chronic renalfailure of all mammals other than those belonging to the FamiliesFelidae and Canidae can also be treated by our method. Needless to say,among the pet animals, the method can be applied not only to thepatients with uremia before receiving dialysis treatment, but also tothe patients with uremia who have already received dialysis treatment.Further, the method may be applied to the patients who have alreadydeveloped a complication due to dialysis treatment. Still further, themethod may be applied to the patients who have already developed theside effects by the active carbon preparation. It should be understood,however, that active carbon has an ability to nonspecifically adsorbsubstances, and the treatment is preferably performed taking the timingand route of administration of the both drugs into consideration.

EXAMPLES

Our agents and methods will now be described more concretely by way ofExamples thereof. However, this disclosure is not restricted to thefollowing Examples.

Example 1: Preparation of Tablets Containing Beraprost Sodium Influenceof Volume and Viscosity of Granulation Solution on Uniformity inContents in Granules and Tablets

As the drug to be administered, beraprost sodium (Sodiumrac-(1R,2R,3aS,8bS)-2,3,3a, 8b-tetrahydro-2-hydroxy-1-[(E)-(3S,4RS)-3-hydroxy-4-methyloct-1-en-6-ynyl]-1H-cyclopenta[b][1]benzofuran-5-butanoate)(BPS) was used. Beraprost sodium, 77.5 parts of base powder (lactose(Pharmatose 200M, OMV Japan)) and crystalline cellulose (Avicel PH-101,Asahi Chemicals) 20.0 parts) were fed into a high speed mixinggranulation machine, and the mixture was stirred for 1 minute. Then apreliminarily prepared aqueous solution for granulation containingberaprost sodium and hydroxypropyl cellulose (HPC-M, Shin-Etsu Chemical)as a binder to a final content of 2 parts was added in a prescribedamount, and mixing granulation was carried out. The obtained granuleswere supplied to a pulverizer and the resultant was dried at 60° C. for10 hours in hot air. The particle size of the resultant was regulatedwith a 20-mesh granulator to obtain dry granules. Magnesium stearate wasadded in an amount of 0.5% based on the dry weight of the granules, andthe resultant was mixed in a V-mixer, followed by tableting with arotary continuous tableting machine using a punch and mortar of 6 mm, 8Rto obtain uncoated tablets with a weight of 80 mg/tablet. The obtaineduncoated tablets were placed in a coating machine, and coating wasperformed spraying a coating solution (Opadry™ (Opadry OYS-9607, JapanColorcon) at 80° C. to obtain tablets coated with a film. The coatingamount was 3 mg/tablet, and the contents of beraprost sodium was 40μg/tablet or 150 μg/tablet.

Example 2 Capsules Containing Beraprost Sodium

The dry granules obtained by the method described in Example 1 werefilled into Qualicaps capsules, Size 5, LOK-CAPS, SHIONOGI) to preparecapsules containing granules. Liquid-containing capsules were preparedby diluting beraprost sodium in polyethylene glycol (Wako PureChemicals) to attain a dose of 10, 30, 70 or 100 μg/28 μl/kg, andfilling the solution into Qualicaps capsules. Beraprostsodium-containing capsules were prepared using a micropipet. Similarly,as a placebo, capsules filled with polyethylene glycol alone in anamount of 28 μl/kg were prepared.

Example 3: Study on Dose of Beraprost Sodium in Healthy Cats

In this study, 3 male cats and 2 female cats, totally 5 Japanese cats,aging 3 to 6 years old were used, which were raised in an animalhospital and for which no abnormalities were observed clinically and byblood test. The subject cats were individually placed in each cage inwhich each cat was allowed to take water ad libitum, and each cat wasfed twice a day after measuring the necessary items. For the purpose ofacclimatization, the cats were measured for blood pressure noninvasivelytwice a day for 2 weeks.

Observations of general clinical symptoms caused by administration ofberaprost sodium to healthy cats were performed as follows: Whetheradverse events such as ataxia, sedation, lying, suffusion of visiblemucosa, lacrimation, hypersalivation, vomiting, diarrhea, incontinenceand change in respiratory conditions were expressed or not was observed.Observation was performed before administration, and 30 minutes, 1 hour,2 hours and 3 hours after each administration during the continuous 7days in which the drug was administered every day. Each evaluation wasperformed by rating in 4 ranks as follows: Respiratory conditions wereranked into: no abnormality observed (Grade 0); accelerated respiration(Grade 1); hypopnea (Grade 2); and dyspnea (Grade 3). Ataxia was rankedinto: no abnormality observed (Grade 0); slight (Grade 1); moderate(Grade 2); and abasia (Grade 3). Sedation (response to verbal contact)and lying (response to verbal contact) were ranked into: no abnormalityobserved (Grade 0); slight (Grade 1); moderate (Grade 2); and noresponse (Grade 3). Evaluations of lacrimation, hypersalivation,vomiting, diarrhea and incontinence were ranked into: no abnormalityobserved (Grade 0); slight (Grade 1); moderate (Grade 2); and severe(Grade 3).

Blood pressure and heart rate were measured as follows: These werenoninvasively measured oscillometrically by using Life Scope 9 producedby NIHON KOHDEN. Systolic pressure, diastolic pressure and heart ratewere measured 5 times each before the administration of beraprost sodiumand 30 minutes, 1 hour, 2 hours and 3 hours after administration ofberaprost sodium at the right foreleg of each cat. From the 5 measuredvalues, the maximum and minimum values were excluded, and the average ofthe remaining 3 measured values was adopted as the measured value of theindividual at the time point. The measurements were carried out everytime for 7 days, and the responses by the respective administrations hadabout the same pattern. In the Example, the results (FIGS. 1a and 1b and2a-2c ) at the first administration of beraprost sodium are shown as arepresentative.

Blood test and blood chemical analysis were carried out as follows:Blood was collected before administration of beraprost sodium and on thenext day of the termination of continuous administration for 7 days ateach dose (the continuous administration of one test compound for 7 daysis hereinafter referred to as “1 series”) at fasting, and analyzed.Inspection items in the blood test were RBC, WBC, PCV, Hb and Plat, andthose in the blood chemical analysis were AST, ALT, T.P, BUN, Cre, Na, Kand Cl.

The administration method was as follows: To each of the 5 healthy cats,4 administration series of beraprost sodium at doses of 10, 30, 70 and100 μg/kg (continuous administration for 1 week at each dose),respectively, and 1 series of placebo which did not contain beraprostsodium were administered sequentially. In each administration series,the same cat was used in each administration series as the subject catfor the correctness of the evaluation of the test drug. To eliminate theinfluence of the test drug of the previous series, wash out period of 2weeks were given after the termination of the respective series. In theberaprost sodium administration series and the placebo administrationseries, the solution-containing capsules prepared in Example 2 wereused. Administration was performed orally at fasting twice a day atintervals of 12 hours for continuous 7 days.

Statistical analysis was carried out using a statistical software StatView J-4.5 (Abacus Concepts). Since the parameter values before andafter the administration to the same individual are compared,significant test was carried out by the paired t-test. In any of thecases, when the significance level was less than 5%, it was judged assignificant. The results are as follows:

As a result of observation of clinical symptoms, adverse events were notobserved at all in the 10 μg/kg/BID and 30 μg/kg/BID series. On theother hand, 2/5 cases in 70 μg/kg/BID, adverse events such as diarrhea,vomiting and sedation of Grade 1 were sporadically observed during 2 to6 days after administration. More particularly, Grade 1 diarrhea wasobserved in cat No. 1 on Day 6 of the continuous administration and incat No. 2 on Days 2 and 5 of the continuous administration. In the 100μg/kg/BID series, in all cats, from Day 2 from the start of theadministration, Grade 2 vomiting, diarrhea and sedation frequentlyoccurred. More particularly, cat No. 1 showed diarrhea on Days 2 and 3of continuous administration; cat No. 2 showed diarrhea on Days 2 and 3and showed diarrhea and vomiting on Day 5; cat No. 3 showed diarrhea andvomiting on Day 2, and diarrhea on Days 3, 4 and 5; cat No. 4 showeddiarrhea and vomiting on Day 2, diarrhea on Day 3, vomiting and loosestools on Day 5, and diarrhea on Day 6; and cat No. 5 showed diarrheaand sedation on Day 2, diarrhea and vomiting on Day 3, and diarrhea onDay 4; within 2 hours from the administration of the respective days ineach case. As for other adverse clinical symptoms, cat No. 5 in 100μg/kg/BID series showed sedation on Day 2 of administration. However,these adverse events disappeared by the next day of finaladministration.

Except for 10 μg/kg/BID series, significant (p<0.05) dose-dependentincrease in heart rate due to beraprost sodium was observed from 30μg/kg/BID, during 30 minutes to 1 hour after the administration ofberaprost sodium (FIG. 1a shows the actual measured values, FIG. 1bshows the rate of change taking the value before administration as100%). The maximum change was about 25% increase after 30 minutes of theadministration of 100 μg/kg. However, with any dose, the heart rate wasrestored to the level before the administration within 2 hours from theadministration. This transient and short-time pattern of increase in theheart rate was the same, either after the first administration or aftercontinuous daily administration of beraprost sodium.

The influence on the blood pressure by the first administration in theadministration series at respective doses is shown in FIGS. 2a-2c . Nosignificant change was observed in any of the systolic pressure,diastolic pressure and pulse pressure. As for the blood cell count,electrolytes, renal function and liver function, no adverse change wasobserved.

Testing methods for the patients showing uremia caused by chronic renalfailure, employed in Example 4 or later, will now be described. Thecriteria and evaluation items for the cats and dogs with chronic renalfailure are as follows: Diagnosis of uremia and chronic renal failurewas performed totally taking clinical findings (including findings ofuremia) and the results of the blood test, blood biochemical analysisand urinalysis into consideration. As the clinical findings of uremia,existence and degree of polydipsia and diuresis, dehydration, anorexia,weight loss, vomiting symptom and state of dehydration were observed.Existence of chronic renal failure was judged based on the serum Crevalue as generally employed. As reference data for grasping the degreeof progress of chronic renal failure, BUN value was also monitored.

In the blood test, RBC, WBC, PCV, Hg and PLT were measured. In the bloodbiochemical analysis, TP, Tcho, AST, ALT, ALP, Tbil, BUN, creatinine,calcium, inorganic phosphorus, glucose, sodium ion, potassium ion andchloride ion were measured. In the urinalysis, protein amount, pH,occult blood, bilirubin and glucose were measured. To eliminate thecircadian rhythm and influence by meals on the values of bloodbiochemical markers, every blood collection was carried out in themorning before feeding (at fasting).

As a more specific criterion for chronic renal failure in cats and dogsin the present Examples, a serum Cre value of 1.4 mg/dL in the bloodtest was employed. Serum Cre value was measured by creatininedeaminase-amidohydrolase method, and the BUN value as reference data forgrasping the degree of progress of chronic renal failure was measured byurease-GLDH method.

This pilot clinical test was performed after obtaining informed consentof the owners of the cats (clients) before the start of the test,wherein the clients received explanations about the fact that beraprostsodium had already been approved as a drug for human chronic arteryobstruction and had already been marketed, and about the safety thereof.Further, giving first priority to the benefit of the patients andclients, conventional therapeutic methods were also combinedappropriately for the individuals with progressed renal failure.Examples of the concomitant drugs employed include synthetic activecarbons such as Covalzin, Kremezin and Nefguard; calcium preparationssuch as calcium carbonate; formula meals such as k/d and low-proteindiets; and infusion solutions. However, when there was a rational reasonand consent by the owner was obtained, beraprost sodium alone wasadministered even to the individual with progressed renal failure. Onthe other hand, drugs thought to influence on the action of beraprostsodium, such as vasodilators (e.g., enalapril), diuretic (e.g.,furosemide), drugs which influence on renal blood flow (e.g., dopamine)and steroids (e.g., prednisolone) were not used.

Administration of beraprost sodium was conducted by orally administeringthe tablet with a content of 150 μg/tablet (Example 1) twice a day (300μg/head/day). The administration was conducted every day and continuedbasically for 6 months, and when the owner desired, the administrationwas continued even after the test period. The Examples include bothcases where the dosing period was less than 6 months and more than 6months.

Evaluation of degree of improvement of the uremic symptoms was carriedout as follows: From the clinical symptoms related to uremia, activityand appetite, which are unlikely to be overlooked and which can beobserved universally from before to after the test period, wereselected. The activity was ranked into: disappeared (score 0); decreased(score 1) and normal (score 2), and the appetite was ranked into: lost(score 0); decreased (score 1); slightly decreased (score 2); and normal(score 3). The evaluation was carried out based on the total points(full points were 5 points). Thus, a higher point indicates moreimprovements in the clinical symptoms.

Example 4: Administration of Beraprost Sodium to Cat with Chronic RenalFailure (Case-A, Administration of Beraprost Sodium Alone, FIGS. 3 a-3c)

Case-A was a male cat of 5 years old, which was diagnosed as chronicrenal failure as a result of overall judgment. After obtaining consentof the owner (client), therapy by administration of beraprost sodiumalone was started. As a result, the score reached 4 in the first monthof the administration of beraprost sodium, and reached 5, the fullpoints, in the second month, and this score was maintained for 6 monthswhich was the dosing period of beraprost sodium. On the other hand, bothof BUN and Cre decreased simultaneously with the start of theadministration of beraprost sodium, and BUN reached the minimum value inthe second month from the start, and serum Cre reached the minimum valuein the fifth month from the start. At the end of the 6-month dosingperiod, there were no differences between the subject and normal cats interms of clinical observations and biochemical markers. Surprisingly,follow up at 6 months after the termination of the administration ofberaprost sodium, that is, at 12 months after the start of theadministration, revealed that not only the clinical conditions were verygood, but also the BUN and serum Cre value were maintained at normallevels. Thus, in this case, not only the uremia, but also the renalfailure itself were completely cured. Although the fact that beraprostsodium restores the once decreased renal function during theadministration thereof so that it enables to “relieve” or “restitute”the subject from renal failure is also a novel discovery, the fact thatthe recovered state is maintained for a long time even after thetermination of the administration of beraprost sodium, that is, “theeffect that renal failure per se is completely cured and the state ismaintained” was totally newly discovered based on this case. During theadministration of beraprost sodium, no adverse event such as diarrhea,vomiting or the like was observed at all.

Example 5: Administration of Beraprost Sodium to Cat with Chronic RenalFailure (Case-B, Administration of Beraprost Sodium Alone, FIGS. 4 a-4c)

The subject patient to whom beraprost sodium was to be administered wasa male Japanese cat of 4 years old with a body weight of 4.0 kg. Thefinding at the first visit was urinary retention due to feline lowerurinary tract syndromes (FUS). In spite of the treatments with adiuretic and antibiotics, only a temporary lull was obtained and theclinical score was continued to be about 3 points for about 1 year.Thereafter, as a result of overall tests, the cat was diagnosed aschronic renal failure. As a result of the administration of beraprostsodium, the total score of the clinical symptoms at 1 month after thestart of administration was increased from 3 points before theadministration to 5 points, the full points, so that amelioration wasobserved. In this case, during the first to second month from the startof the administration of beraprost sodium, slight amelioration of theclinical symptoms was observed, but there was a tendency that BUN valueand serum Cre value increase. However, surprisingly, from the fourthmonth from the start of the administration of beraprost sodium, BUNvalue and serum Cre value sharply decreased to the normal values, andthe clinical score reached 5 points, the full points. In this case,apparent correlation between the amelioration of clinical symptoms andthe decrease in the values of the markers of renal function wasobserved. With the amelioration of the uremic symptoms, body weight wasincreased. In response to the strong demand by the owner (client), thedaily administration of beraprost sodium has continued for 1 and halfyears, but no adverse event has been observed at all, and Cre and BUNare maintained at the normal levels. Thus, it was proved that beraprostsodium can be administered safely for a long time. Further, beraprostsodium not only has an excellent effect to ameliorate uremia, but alsoit has an effect which is clearly different from the conventionalconcept of the amelioration of chronic renal failure, which conventionalconcept is to suppress or stop the progress of the aggravation of renalfunction. That is, the totally new effect of beraprost sodium whichrestores the once decreased renal function during the administrationthereof so that it enables to “relieve” or “restitute” the subject fromrenal failure was also discovered in this case too. In this case too,during the administration of beraprost sodium, no adverse event such asdiarrhea, vomiting or the like was observed at all.

Example 6: Administration of Beraprost Sodium to Cat with Chronic RenalFailure (Case-C, Administration of Beraprost Sodium Alone, FIGS. 5 a-5c)

This case was a female cat of 16 years old which was diagnosed aschronic renal failure. Progressed uremia concurred so that it was a casefor which other drugs were to be used concomitantly if a conventionaltherapy was to be applied. However, after extensive explanation to andconsent by the owner (client), beraprost sodium alone was administered.At the second week from the start of the administration of beraprostsodium, the clinical score was largely improved from 2 points to 4points. Although the clinical score was again decreased to 2 points atthe sixth week, thereafter, the clinical score was 4 points, and after2.5 months, the clinical score reached 5 points, the full points. Duringthis period, the serum Cre value did not change or slightly increased,but BUN value continuously increased. By this Example, it was provedthat beraprost sodium has an effect to ameliorate uremia even under thecondition that the renal failure was not improved in terms of values. Inthis case too, during the administration of beraprost sodium, no adverseevent such as diarrhea, vomiting or the like was observed at all.

Example 7: Administration of Beraprost Sodium to Cat with Chronic RenalFailure (Case-D, Administration of Beraprost Sodium Alone, FIGS. 6 a-6c)

The subject patient to whom beraprost sodium was to be administered wasa male Siamese cat of 17 years old with a body weight of 4.5 kg.Although it was a progressed case, based on the overall judgment,beraprost sodium alone was administered without any conventional therapysuch as using a concomitant drug or infusion solution. At one monthbefore the start of administration of beraprost sodium, both appetiteand stamina were unstable and the score was 2 points. However, as aresult of administration of beraprost sodium, the clinical total scoreafter one month was drastically improved from 2 points beforeadministration to 5 points, and the body weight was also increased. Inthis case too, during the administration of beraprost sodium, no adverseaction causing diarrhea, vomiting or the like was observed at all. Onthe other hand, BUN value reflecting the renal function was continuouslyincreased even after administration of beraprost sodium. Serum Cre valuealso kept a high value. In this case too, the effect of beraprost sodiumto ameliorate uremia by beraprost sodium per se was clearly observedeven though the markers of renal function were not improved. In thiscase too, during the administration of beraprost sodium, no adverseevent such as diarrhea, vomiting or the like was observed at all.

Example 8: Administration of Beraprost Sodium to Cat with Chronic RenalFailure (Case-E, Combination Therapy, FIGS. 7 a-7 c)

The subject patient to whom beraprost sodium was to be administered wasa male Japanese cat of 14 years old with a body weight of 3.5 kg. Fromabout 10 years ago, urinary retention and cystitis due to lower urinarytract syndromes (FUS) repeatedly occurred. From several months ago,anorexia and emaciation became prominent, and the cat was diagnosed asrenal failure complicated with severe uremia. From several months ago,the cat was treated with 200 mg/BID of Kremezin, diuretic, infusionsolution, calcium carbonate preparation and formula meal (k/d meal), butthe uremia shown by vomiting, anorexia and loss of activity was notimproved at all, and constipation due to the active carbon preparationwas also induced. In response to this, Primperan Syrup (FujisawaPharmaceutical, general name: metoclopramide) was administered, but theclinical score became 0. In view of this, after obtaining consent by theowner, administration of beraprost sodium was started in addition to theconventional therapies which were treatments with 200 mg/BID ofCovalzin, 200 mg/BID of a calcium preparation, formulation meal andinfusion solution. The clinical total score after one month from thestart of the administration of beraprost sodium was largely increased to4 points from 0 point before the administration. Thereafter, althoughthere was a temporary decrease in the score, the score was 5 points, thefull points, for about 3 months, and the uremic symptoms were largelyimproved. However, according to the evaluation of renal function duringthis period, serum Cre value was kept high, and BUN value evencontinuously increased, so that conditions opposite to the drasticimprovement in the uremic symptoms were observed. Since dehydrationcompletely disappeared, infusion became not necessary during thisperiod. Since constipation which is a side effect of Covalzin becamesevere again 3 months after the start of the treatment, only theadministration of Covalzin was eliminated in the fourth months from thecombination therapy. From this time point, a low clinical score of 3points continued for about 2 months, but by the combination therapyincluding beraprost sodium, the clinical score was restored to 5 pointswhich were the full points from the 7th month to 8th month, so that theuremic symptoms were drastically improved. Surprisingly, at this timepoint at which the uremic symptoms had been improved, the Cre value waskept high, and BUN value was even largely increased. After stopping theadministration of Covalzin, constipation completely disappeared. On theother hand, although administration of beraprost sodium was continued,no adverse events in the digestive tract such as vomiting and diarrheawere not observed at all thereafter. By this, the excellent effect ofberaprost sodium to ameliorate uremic symptoms was additionallyconfirmed clearly in spite of the fact that Cre and BUN which are therenal function markers showing renal failure were still continuouslyincreased. In this case too, during the administration of beraprostsodium, no adverse event such as diarrhea, vomiting or the like wasobserved at all.

Example 9: Administration of Beraprost Sodium to Cat with Renal Failure(Case-F, Diabetic Nephropathy, FIGS. 8 a-8 c)

The patient was a male cat of 11 years old, was diagnosed as chronicrenal failure derived from diabetic nephropathy from the past historyand the values obtained by blood biochemical analysis, and showed uremicsymptoms. First, for 2 months, insulin (protamine zinc insulin, 2units/BID) for treating diabetes, and a calcium carbonate preparationand an active carbon preparation for uremia were administered. As aresult, as for diabetes, the fasting blood glucose became 100 mg/dL, andthe urine sugar was stabilized to ±. However, as for renal failurecondition, the BUN value was invariably high, and the uremic symptomswere not improved and the clinical score remained to be 2 to 3 points.In view of this, daily administration of beraprost sodium was started inaddition to the insulin therapy and combination therapy with otherdrugs. As early as in the first month after the start of theadministration of beraprost sodium, the clinical symptoms weredrastically improved to the score of 5 points which were the fullpoints, and the uremic symptoms disappeared. For 10 months thereafter,this condition continues. On the other hand, BUN value which is a renalfailure marker was kept high even after the start of the administrationof beraprost sodium, and serum Cre value continuously increasedthroughout the entire period. By this case, it was proved that beraprostsodium can be safely and effectively used for the therapy of uremia inpatients with chronic renal failure whose primary disease is diabetes.Further, it was additionally confirmed actually that beraprost sodiumhas an effect to ameliorate uremic symptoms even if the renal failureitself is not improved. In this case too, during the administration ofberaprost sodium, no adverse event such as diarrhea, vomiting or thelike was observed at all.

Example 10: Amelioration of Uremic Symptoms by Tablets with LowBeraprost Sodium Content (Case-G, Administration of Beraprost SodiumTablets Alone)

The cat with chronic renal failure used in this Example was a cat keptand raised as a transfusion donor for surgeries. By virtue ofvaccination at appropriate timings, the cat was free from past historyof infectious diseases, and the health condition had been periodicallyfollowed by various blood tests. However, by overall judgment, the catwas diagnosed as having transferred to chronic renal failure, andadministration of beraprost sodium was started. Since the effectiveness(anti-uremia effect) of administration of beraprost sodium at a dose of150 μg/head/BID had been confirmed by the information on the previouslystudied clinical cases, a lower dose of 40 μg/head/BID (Example 1) wasadministered to this case (case-G), and the tests were carried out every2 weeks. Before the administration of beraprost sodium, BUN value was52.2 mg/dL, and the serum Cre value was 2.56 mg/dL. After starting theadministration of beraprost sodium at a dose of 40 μg/head/BID, BUNvalue was almost kept constant, and Cre continuously increased to 2.79mg/dL on Day 28, and to 3.02 mg/dL on Day 56. On the other hand,increase in body weight was observed from an early stage after the startof the administration of beraprost sodium, and the body weight wasincreased from 3.2 kg before the administration of beraprost sodium to3.83 kg on Day 14 and to 3.95 kg on Day 56. Restitution from uremicsymptoms was also observed from an early stage, and the score reached 5points which were the full points as early as on Day 14 from 3 pointsbefore the administration of beraprost sodium, and the full points arekept thereafter up to Day 56. Thus, by this Example too, the effect ofameliorating uremic symptoms per se accompanied by chronic renal failureby the administration of beraprost sodium at a lower dose was alsoconfirmed in the condition that the serum Cre value was continuouslyworsened. In this case too, during the administration of beraprostsodium, no adverse event such as diarrhea, vomiting or the like wasobserved at all.

Example 11: Increase in Body Weight by Administration of BeraprostSodium

Administration of beraprost sodium clearly improved appetite and desirefor water, and increased body weight. The cases thereof are shown inTable 1. As is apparent from the table, by the administration ofberaprost sodium, amelioration and disappearance of uremic symptomsshown by restoration of body weight expressed by an objective value, inaddition to the results of the visual observations such as improvementin activity and increase in appetite, gave great satisfaction to theowner not to mention the patient cat.

TABLE 1 Changes in Body Weight of Cats with Renal Failure duringAdministration of Beraprost Sodium Changes in Body Weight duringAdministration of Beraprost Sodium (kg, month) Case Age −2 −1 0 1 2 3 45 6 Case-A 5 5.2 5.5 5.5 5.5 5.7 5.7 6.0 Case-B 4 3.5 3.6 3.6 3.7 3.94.0 4.0 Case-C 16 2.8 2.7 2.8 2.9 2.9 2.9 3.0 3.0 Case-D 17 4.5 4.5 4.54.9 4.9 4.9 4.9 Case-E 14 3.5 3.8 3.7 3.6 3.7 3.7 3.6 Case-F 11 4.7 5.05.0 5.5 5.5 5.7 5.8

Example 12: Administration of Beraprost Sodium to Dog with Renal Failure(Case-H, FIGS. 9 a-9 c)

The subject patient to whom beraprost sodium was to be administered wasa female dog (hybrid) of 14 years old with a body weight of 6.4 kg. Thepatient visited the hospital with chief complaints of anorexia anddrastic decrease in activity. Based on the past history, clinicalobservations and results of clinical test, the dog was diagnosed aschronic renal failure. The clinical score at the first visit was as lowas 2 points, and treatment with Covalzin at a dose of 200 mg/BID wasstarted. As a result, although the clinical score was little bitimproved to 3 points in 2 weeks, thereafter, the low value of 3 pointscontinued for 2 months, and no more improvement was observed. In view ofthis, after obtaining consent by the client, treatment with Covalzin wasstopped, and switched to administration of beraprost sodium alone.Beraprost sodium was diluted with polyethylene glycol (Wako) to attain adose of 150 μg/capsule, and was divided into capsules (size 5 ShionogiQualicaps). Beraprost sodium was orally administered twice a day atfasting. As a result, as shown in FIGS. 9a-9c , the clinical score wasincreased to 4 points in the first month from the start ofadministration, and reached 5 points which were the full points in thesecond month. On the other hand, BUN which is a renal failure marker didnot almost change, and serum Cre value increased in spite of the factthat improvements in the clinical symptoms were drastic after the startof the administration of beraprost sodium. This phenomenon that theimprovements in the clinical symptoms and the values of renal failuremarkers do not necessarily correlate is the same as in cats with renalfailure. In the above-described series of therapy, feeding of a formulameal and infusion were not performed at all. In this case too, duringadministration of beraprost sodium, no adverse event such as diarrhea,vomiting or the like was observed at all. Based on the above, it wasfirst discovered that in chronic renal failure of dogs, in addition tocats, the effect of beraprost sodium to ameliorate uremic symptoms isclearly shown in spite of the fact that Serum Cre value and BUN valuewhich are markers of renal function indicative of renal failure arecontinuously worsened.

What is claimed is:
 1. A method of ameliorating symptoms of uremiacomprising administering a therapeutic agent comprising beraprost oreach isomer constituting beraprost, or a salt thereof as an effectiveingredient to an animal belonging to the Family Canidae, wherein aneffective dosage is a dosage that does not significantly lower bloodpressure.
 2. The method according to claim 1, wherein the dose peradministration is not more than 100 μg/kg.
 3. The method according toclaim 1, wherein the dose per administration is 40 to 150 μg/body andwherein the dose per administration is not more than 100 μg/kg.
 4. Themethod according to claim 1, wherein the dose per administration is 12.5to 55.6 μg/kg.
 5. The method according to claim 1, wherein the dose peradministration is about 23 μg/kg.
 6. The method according to claim 1,wherein the dose per day is 80 to 300 μg/body and the dose peradministration is not more than 100 μg/kg.
 7. The method according toclaim 1, wherein the dose per day is 25.0 to 111.2 μg/kg and the doseper administration is not more than 100 μg/kg.
 8. The method accordingto claim 1, wherein the dose per day is about 46 μg/kg.
 9. The methodaccording to claim 1, wherein the number of dose administrations istwice daily.